Effect of temperature on the life cycle of Harmonia axyridis (Pallas), and its predation rate on the Spodoptera litura (Fabricius) eggs

Biological control is one of the strategies of pest control which is determined by the biological fitness and metabolic rates of the predator species used. Temperature and resource are important factors which influence the role of insects as biocontrol agents. Harmonia axyridis is a cosmopolitan and non-specific polyphagous predator. It can survive ecologically diverse environments and exploit multiple preys. This study investigated the effects of temperature on the population parameters of H. axyridis and its predation on the eggs of prey Spodoptera litura. For this purpose, an age–stage, two-sex life table of the predator was constructed at four constant temperatures, i.e. 15, 20, 25 and 30 °C, under laboratory settings of: 70 ± 5% RH, and 16:8 h (L: D) photoperiod. A computer simulation was then used to project the population and predation responses with respect to temperatures tested. We found that the development of larvae and adult (male/female) stages of H. axyridis decreased with colder temperatures (i.e., 15 and 20 °C) but increased with warmer temperatures (25 and 30 °C). The intrinsic rate of increase (r) and mean generation time (T) were 0.0662 d−1 and 79.84 d at 15 °C, 0.0843 d−1 and 64.90 d at 20 °C, 0.1067 d−1 and 48.89 d at 25 °C, and 0.1378 d−1 and 35.55 d at 30 °C, respectively. The mean duration of the total pre-adult stage was 44.26, 32.91, 20.63, and 15.39 d at 15, 20, 25, and 30 °C, respectively. At 30 °C. the finite rate of increase (1.1477 d−1) was the highest and the mean generation time (35.55 d) was the shortest. The net predation rate (C0) was 7935.54, 10,466.28, 10,139.38, and 7126.36 eggs at 15, 20, 25, and 30 °C, respectively. Population and predation projections were proportional to temperature. These findings are important for modelling the population responses of H. axyridis to climate change and tailoring integrated pest management strategies to altered climates.


Materials and methods
Mass rearing of insects. Harmonia axyridis population culture was established from a laboratory reared stock at Key Laboratory of Huazhong Agricultural University (HZAU), Wuhan, China, during February 2019. The adult insects were grouped in transparent plastic boxes (12 cm × 16 cm × 7 cm) with moistened cotton swabs, and reared on ad libitum diet of Acyrthosiphon pisum nymphs Harris (Hemiptera: Aphididae). A. pisum was reared on faba bean Vicia faba L. plants under conditions of 22 ± 1 °C, 65 ± 5% RH and 16:8 (L:D) h photoperiod. The seeds of faba bean were purchased online and grown inside cages. A. pisum was then introduced to these faba beans to start an aphid laboratory culture. Eggs of H. axyridis were removed daily and kept in 9 cm Petri dishes on moistened tissue paper. Hatchlings were transferred to separate Petri dishes to avoid cannibalism. These Petri dishes had folded tissue paper, A. pisum nymphs as food and moist cotton as water source. All the insects were kept under controlled laboratory conditions at 70 ± 5% RH, 24 ± 1 °C, and 16:8 (L:D) h photoperiod. All larval, pupal, and adult stages were maintained under same settings as above. The plant materials used were obtained with prior permission, and the present study complies with relevant guidelines and legislation.
The pupae of S. litura were purchased online from Henan Jiyuan Baiyun Industrial Co., Ltd, China, in September 2018 and placed in a transparent glass jar (20 cm × 12 cm × 8 cm) and incubated inside the growth chamber (Jiangnan Instruments RXZ-430, Ningbo, China) at settings of 26 ± 1 °C and 70 ± 5% RH under a 16:8 h photoperiod (L: D) to obtain adult populations. Hatchlings from S. litura eggs were fed with an artificial diet and cultured until the reach of final instar stage inside a mesh covered transparent circular glass jars (1 L). The artificial diet prepared followed Saljoqi, et al. 37 . The artificial diet (semi-solid) consisted of yeast powder (24 g), ascorbic acid (2.35 g), methyl-4-hydroxy benzoate (1.5 g), distilled water 550 mL, kidney bean flour (150 g), agar (8.4 g), sorbic acid (0.75 g), formaldehyde solution (1 mL) and streptomycin (0.75 g). The final caterpillars were isolated and subsequently cultured until maturity inside similar jars with 20% glucose solution as adult's diet and clean pieces of paper covering jar's bottom as oviposition substratum. The cotton wool strips (2 cm wide, 7-12 cm long) were placed in jars as a suitable oviposition substrate in order to obtain new oviposition. Oviposition was checked daily. Hatchlings were reared together in a separate Petri dish. Same age larvae were reared together using the same artificial diet as above to maintain population homogeneity.
Life table and predation trials. The experimental setup used here was a Petri dish (6 cm), and one leaf disc of cotton (Gossypium hirsutum L. cv. Varamin) placed on a 0.5 cm thick layer of 1.5% agar to fix the leaf disk. Paired H. axyridis adults were separated from the stock culture and reared with S. litura eggs in transparent plastic cups (9 cm in diameter and 7 cm in height) covered with fine nylon netting for ventilation under  38 . Experimental cohorts were generated as fifty individuals per temperature. Survival rate and time to hatch were recorded for each stage in each temperature treatment. The number of surviving larvae and the total developmental period from larva to pupa were also recorded. Predation was recorded for both larval and adult stages of the predator. After eclosion, the predator larvae were fed with S. litura eggs until pupation. In addition to this, male and female adults that emerged from the pupae on the same day from a given temperature were numbered and coupled in the insect boxes. Adult pairs were monitored to ensure that mating occurred, and time allowed for mating event was ~ 10 h a day, and no prey was made available during mating events throughout the trial duration. One or a few matings are reported sufficient for coccinellids females to fertilize all eggs. No eggs provision during mating events was meant to avoid any potential disturbance during this activity. After mating, the pairs were separated and shifted cautiously to their respective Petri dishes with eggs. The life span of an adult male is shorter than female, hence extra pairs of insects were raised in parallel to supply supplementary males for mating depending on the need at each temperature, simultaneously. The number of eggs laid per female including the cannibalized ones and adult mortality was noted twice daily until all female adults died. About 400-500 eggs of S. litura were supplied daily to the larvae and adults on cotton leaves. The counted numbers of S. litura eggs were fed to each stage of the predator. Predation data were recorded daily as numbers of eggsdamaged or consumed. Petri dishes and boxes were cleaned daily with 75% ethanol.
Data analysis. The data were analyzed by using TWOSEX-MSChart 39 according to age-stage, two-sex life table theory 14,40 . The age stage-specific survival rate (s xj ), age-specific survival rate (l x ), age-specific fecundity (m x ), the net maternity (l x m x ), age-stage life expectancy (e xj ), age-stage reproductive value (v xj ), population and reproduction parameters were calculated accordingly. All oviposited eggs per female were counted to calculate the fecundity. The age-stage survival rate s xj was calculated as: where n 01 is the number of individuals used at the beginning of the life table study and n xj is the number of individuals surviving to age x and stage j 41 . The age-specific survival rate (l x ) was calculated as: and age-specific fecundity (m x ) were obtained from the following formula, where β is the number of stage s xj is the age-stage specific survival rate, i.e. the possibility that a new insect (newly hatched individual) will live or exist to age x and stage j.
The net reproductive rate (R 0 ) is defined as the total number of offspring that an insect or individual can produce during its lifetime and was calculated as: The intrinsic rate of increase (r) was then predicted iteratively following the Euler-Lotka equation with age indexed from 0 42 by the following formula, The finite rate of increase (λ) was calculated as: The mean generation time (T) is the time duration that a population needs to increase to R 0 -fold of its size at the stable age-stage distribution, and was calculated as: where s ′ iy is the probability that an individual of age x and stage j will survive to age i and stage y and it is calculated by assuming s xj = 1.
The age-stage reproductive value (v xj ) was defined as the contribution of individuals of age x and stage j to the future population and is calculated according to Tuan et al. 44,45 .
Raw data on daily predation rates were analyzed according to 46 by CONSUME-MSChart 47 . The age-specific predation rate (k x , the number of prey consumed by the surviving individuals at age x), age-stage specific predation rate (c xj , the mean predation rate of individuals at age x and stage j), age-specific net predation rate (q x ), net predation rate (C 0 ), transformation rate (Q p ), finite predation rate (ω) and stable predation rate (ψ) were calculated.
The age-stage specific predation rate c xj was calculated as: where d xj,i is the calculated predation rate for the ith individual at age x and stage j. The age specific predation rate (k x ) was calculated as: The age specific net predation rate (q x ) by considering survival rate was calculated as: The net predation rate (C 0 ) is defined as the number of prey consumed by an average individual predator during its lifetime. It includes all individuals that died in the preadult stages and those that survived to the adult stage. It was calculated as: The transformation rate (Q p ) is the number of prey needed for a predator to produce a single offspring. It was calculated as: The finite predation rate (ω) was calculated according to Yu, et al. 30

as:
where a xj is the proportion of individuals belonging to age x and stage j in a stable age-stage distribution, ψ is the stable predation rate.
Variances and standard errors (SE) of all parameters were determined by using 100,000 bootstraps replications [48][49][50] . We used paired bootstrap tests to compare differences among the temperatures at 5% significance level based on the confidence interval of difference 51 . All graphs were plotted with SigmaPlot (ver. 12, Systat Software, Palo Alto, CA). Harmonia axyridis at all temperatures and it was projected for 90 d to obtain the total population size assuming a scenario without suppression by biotic and/or abiotic factors and predation potential with unlimited prey, i.e., S. litura eggs. The total population size at time t was calculated as: where n xj,t is the number of individuals of age x and stage j at time t 52 .
The predation potential at time t was calculated following Huang, et al. 52 : To reveal the variability of both projections, we sorted the 100,000 bootstrap results of the finite rate of increase (λ) to find the 2.5th and 97.5th percentiles, i.e., the 2,500th and 97,500th sorted bootstrap samples. We then used the bootstrap life table samples that generated the 2.5th and 97.5th percentiles of the finite rate of increase (λ) to project the population to represent the confidence interval of the projected populations 52 . Projections were made based on the method described by 41,53 and via TIMING-MSChart computer program 54 .  Reproduction and population parameters of H. axyridis. The temperature effects were assessed on the estimated intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R 0 ), and mean generation time (T) for H. axyridis feeding on S. litura eggs by applying the bootstrap technique with 100,000 resamplings (Table 2). Population parameters i.e. r, λ, R 0, and T of H. axyridis on S. litura eggs under influence of temperature are presented in Table 2. The r (the intrinsic rate of increase) and λ (finite rate of increase) highest      Table 3). The age-stage specific predation rate (c xj ) of H. axyridis which means the mean predation rate of individuals at age x and stage j was presented in Supplementary Fig. S4. While the long survival durations increase total predations in lower temperatures i.e. 15 and 20 °C, daily predation through the stages was generally higher at 25 and 30 °C. The net predation rate (C 0 ) was greatest i.e., 10,466.28 and 10,139.38 at 20 and 25 °C than those at 15 and 30 °C with 7935.54 and 7126.36 eggs/individual, respectively (Table 4). There was no significant difference in transformation rate (Q p ) at all tested temperatures. The transformation rate (Q p ) was highest at 25 °C, which indicates that H. axyridis requires 55.05 S. litura eggs for the production of each egg at 20 °C. Stable and finite predation rates differed among all tested temperatures. The highest values for both parameters were obtained at 30 °C, whereas lowest values were at 15 °C. Table 2. Reproduction and population parameters (Mean ± SE) of Harmonia axyridis fed on Spodoptera litura eggs at four different temperatures. Standard errors were estimated by using the bootstrap technique with 100,000 resampling. Difference was compared using the paired bootstrap test (P < 0.05). The means within a row followed by a different lowercase letters indicate significant differences among the temperatures. APOP adult preoviposition period, TPOP total preoviposition period. a R 0 = net reproductive rate; r = intrinsic rate of increase; λ = finite rate of increase; T = mean generation time; F = fecundity; O d = oviposition days. www.nature.com/scientificreports/ Age-specific predation rate (k x ), age-specific net predation rate (q x ) cumulative net predation rate (C 0 ) for H. axyridis feeding on S. litura eggs at four different temperatures were presented in Fig. 2. The k x gives the average number of S. litura eggs eaten by H. axyridis of age x, while q x gives the weighted number of aphids eaten by H. axyridis of age x. As H. axyridis feed with S. litura eggs only in the larval and adult period, the increase in k x and q x was linked to survival rates until the adult period with a stepwise reduction beginning from the adult period. There were two gaps in the curves for k x and q x at all tested temperatures due to non-predator i.e., egg and pupa Population and predation projection. Both the population and predation projection for 90 days began with 10 H. axyridis pairs at all temperatures (Fig. 3). The variability of both population growth and predation potential was assessed by utilizing the life tables that generated the 0.025th and 0.975th percentiles of λ of 100,000 bootstrap results. The total population size of H. axyridis was highest at 30 °C, projected to exceed 8.9 × 10 6 individuals, whereas the population at 15 °C was estimated at 4.3 × 10 4 individuals. The total population at 25 and 30 °C increased significantly than those at 15 and 20 °C. At lower temperatures (i.e., 15 and 20 °C), the curves of different stages tended to be straight lines (Fig. 3). Total consumption of S. litura eggs was highest and projected to reach 5.24 × 10 8 , while egg consumption was approximately 4.9 × 10 5 at 15 °C after 90 days (Fig. 3).

Discussion
Coccinellid species are generalist predators and the type of prey they consume shows great impacts on their fitness and predation. The amount and quality of food is very important because it influences directly biological aspects of predators 55 . When ingested food is scarce or inferior quality, the development time usually increases and the reproductive rate, i.e., oviposition, fecundity and fertility decreases 56 . Given the relevance of coccinellids for biological control, much attention has been paid to documenting feeding habits within this group. Food that provides better development and reproduction is considered essential for coccinellids. Many aphids, psyllids and mealybugs are important preys. Many other energy sources as preys that prolong survival are characterized as alternative 57 . Coccinellidae are shown to reproduce even more on non-aphid preys in some studies, which demonstrates the importance of alternative resource 58,59 . Eggs of lepidopterans were the best diet for H. axridis because shorter duration and higher survival of the larval stage of this predator was evident on the eggs 34,60 . Abdel-Salam and Abdel-Baky 34 estimated the R 0 of H. axyridis when feeding on frozen and fresh eggs of the Sitotrega cerealella, and showed moth eggs to be a better diet based on findings obtained by applying the female age-specific life table and adult age. This shows that scales and chorion of eggs were not barriers for larval as well as adults feeding, as reported for H. axyridis with S. litura eggs 35 . The current study is the first reporting on the survivorship, development, reproduction, and longevity of H. axyridis on S. litura eggs at different temperatures. Table 3. Stage daily predation (D j ) (preys/individual) (Mean ± SE) of Harmonia axyridis fed on Spodoptera litura eggs at four different temperatures. Standard errors were estimated by using the bootstrap technique with 100,000 resampling. Difference was compared using the paired bootstrap test (P < 0.05). The means within a row followed by a different lowercase letters indicate significant differences among the temperatures, while different uppercase letters within the same column indicate significant differences between female and male adult stage daily predation. We also used computer projections based on bootstrap percentile confidence intervals to better understand the variability of population growth and predation parameters. Age-specific survival rate (l x ), age-specific predation rate (k x ), age-specific net predation rate (q x ) cumulative net predation rate (C 0 ) of Harmonia axyridis feeding on Spodoptera litura eggs at different temperatures.    27 reported that H. axyridis fed with C. atlantica showed the greatest fecundity at 15 °C (i.e., 805.7 ± 127.3) than when fed on the same prey at 20 and 25 °C (i.e., 608.5 ± 113.7 and 614 ± 129.2, respectively). In our experiment, the fecundity comparatively reduced at higher temperatures but not the predation that increased with warmer temperatures. Higher temperatures can reduce reproductive fitness of H. axyridis, and other researchers have reported similar findings 62 , which means H. axyridis survives thermally complex and diverse environments with such a trade off. This research was conducted using total fecundity without the exclusion of unhatched eggs, however it was showed by Mou, et al. 63 that the life table and predation parameters differ depending on whether the egg viability factor is included or excluded. As coccinellid's eggs are not always fertile, and as that fertility rate varies with temperature and predator diet, with resulting consequences on biological procedures 27 , other investigations should look at fertility as well as fecundity to gain a better understanding of this predator's biological performance. The duration of adult pre-oviposition days including APOP and TPOP was shortened with higher temperatures. These results are in agreement with those of other researchers such as Castro, et al. 27 , who reported the highest and the lowest APOP and TPOP for H. axyridis when it was fed with C. atlantica at 15 °C (6.10 and 82.40 d) and 25 °C (5.80 and 76.87 d), respectively.
We found that predation of H. axyridis was also temperature-dependent and increased with warmer temperature. Warming is expected to expedite predation because it boosts the predator's capacity to grab and handle prey in response to increased metabolic rate that can result from increased development and fostered growth 12 . In comparison to first three instars, the fourth larval instar and female H. axyridis consumed more S. litura eggs and showed the maximum predation capacity at these stages. Other researchers have reported that the predation rate of preadult stages of H. axyridis generally increased with larval sizes 12,35 . Females consumed more eggs than did males because females need to oviposit. The sexes differed in our findings in a similar way. The female age-specific fecundity correlates with the trend of predation rate. Similar results have also been reported by another researcher who showed fourth instar of Hippodamia variegata (Coleoptera: Coccinellidae) to consume significantly more Aphis fabae Scopoli (Hemiptera: Aphididae) than other stages of the predator at 23 °C 64 .
This study concludes that H. axyridis development depends on the temperature and predation varies with predator stage, temperature, and resource being consumed. On the eggs of S. litura, longevity , reproductive rate and mean generation time of H. axyridis were much improved at 15 and 25 °C than at 25 and 30 °C. Lower temperatures can thus be favorable for mass rearing the predator. Warmer temperatures (30 °C) can lead to an accelerated development and growth of this predator, which means an increased predation rate. These findings may help with devising effective biocontrol programs against S. litura using H. axyridis as the predator, probably by identifying potential predator stage and optimal release time. This study further provides useful information about the future consequences of warming on this predator development and predation parameters, which can help attune bio-based IPM programs according to changing climates 11,12. Hance, et al. 65 reported that divergences between thermal optimums of predators and preys may promote disturbance of geographical or temporal synchronization, increasing the risk of pest outbreaks. Temperature limits pest distribution; however, warming has resulted in more suitable disciplines for pests, e.g., insect range expansions. Our experiments used a thermal maximum of 30 °C, and temperatures in Asia can reach as high as > 40 °C for short periods of time at midday during hot summer 66 . Presumably, S. litura in this region is locally adapted to short bouts of high heat and can withstand it, although additional research is needed to confirm that host eggs exposed to high heat are suitable for predator development. Although the present study was performed under laboratory conditions, it may still provide information that can complement field studies. Future field experiments under controlled conditions are recommended to provide a much realistic view of the effectiveness of H. axyridis in the field against this serious pest.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.